Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine involved in the production and function of hematopoietic cells, and GM-CSF plays in particular a major role in responses to infection and physiological and pathological inflammatory processes. GM-CSF is produced in many cell types, and increases in the intracellular Ca2+ concentration are, like in many other systems, of major importance in the intracellular signaling that determines GM-CSF expression after receptor stimulation of the cells. Previous studies have shown that the Ca2+/calmodulin-dependent phosphatase calcineurin (CN) mediates stimulation of GM-CSF transcription in response to Ca2+. This thesis shows that Ca2+ signaling also regulates GM-CSF transcription negatively through Ca2+/calmodulin-dependent kinase II (CaMK II) phosphorylation of serines in the autoinhibitory domain for DNA binding of the transcription factor Ets1. Mutation of the CaMK II target serines increased transactivation of the GM-CSF promoter/enhancer and decreased the sensitivity to inhibition by increased Ca2+ or constitutively active CaMK II. The Ca2+-dependent phosphorylation of Ets1 was also shown to reduce the binding of Ets1 to the GM-CSF promoter in vivo. RUNX1, also known as acute myeloid leukemia 1 (AML1), is one of three mammalian RUNX transcription factors and has many essential functions in hematopoiesis. RUNX1 has also many important roles in the immune system, and RUNX1 is the most frequent target for chromosomal translocation of genes in acute human leukemias. This thesis shows that RUNX1 directly interacts with both subunits of CN and that the strongest interaction is localised to the regulatory CN subunit and the DNA binding domain of the RUNX protein. Constitutively active CN was shown to activate the promoter/enhancer of GM-CSF synergistically with RUNX1, RUNX2 or RUNX3, and the Ets1 binding site of the promoter was shown to be essential for the synergy between RUNX1 and CN in Jurkat T cells. The analysis suggests that Ets1 phosphorylated by the protein kinase glycogen synthase kinase-3β is the target of RUNX1-recruited CN phosphatase at the GM-CSF promoter. Transforming growth factor-β (TGF-β) is another multipotent cytokine that often has a role opposite to that of GM-CSF in inflammatory responses since it is a potent suppressor of immune cells and therefore is anti-inflammatory. This thesis shows that TGF-β can decrease transcription from a GM-CSF promoter/enhancer. Certain constitutively active TGF-β receptors and the TGF-β activated transcription factor Smad3 could also repress GM-CSF transcription, whereas several other Smad proteins did not have this inhibitory effect. The inhibition required intact DNA binding ability of Smad3, and the 125 bp upstream of the transcription initiation site, which was sufficient for the inhibition, contains several weak Smad binding sites near the TATA box next to an Ets1 site of the promoter. Smad3 was able to bind to the promoter DNA together with Ets1 and could also be in complex with Ets1 in the absence of DNA. Surface plasmon resonance analysis revealed that Ets1 interacted with the DNA binding domain of Smad3, and the binding constant of this interaction was about 1 µM. The results identify a negative regulation of the GM-CSF promoter by TGF-β signaling through direct Smad3 binding and indicate that the mechanism is by Smad3 interaction with Ets1 and perhaps other proteins around the TATA box of the promoter. This thesis also identifies a novel transactivation domain in the N-terminal of RUNX1 including the N-terminal α-helix in the DNA binding domain. The domain was also required for RUNX2 and RUNX3 transactivation. Despite this, the N-terminal domain of RUNX1 was not essential for RUNX1 function in megakaryocytopoiesis in vitro from mouse embryonic stem cells.

Systemic lupus erythematosus (SLE) is an autoimmune, inflammatory disease characterized by autoantibody production and an activated type I interferon system. Cardiovascular disease (CVD) is as a major cause of morbidity and mortality. The aim of this thesis was to identify genetic risk factors for CVD in SLE. The role of T cells in regulation of the interferon-α (IFNα) production by plasmacytoid dendritic cells (pDCs) was also investigated.    In paper I, a thicker intima, thinner media and increased intima/media ratio was found in young premenopausal women with SLE compared to healthy controls indicating increased cardiovascular risk. As traditional ultrasound assessment of the common carotid intima-media thickness (CCA-IMT) in SLE has given conflicting results separate measurement of the intima and media can be a useful tool to identify SLE patients at increased risk of CVD.    In paper II, an association was demonstrated in SLE between a STAT4 risk allele and ischemic cerebrovascular disease and presence of anti-phospholipid antibodies (aPL). The association remained after adjustment for traditional CVD risk factors. A possible mechanism for this association is that the risk allele leads to increased production of aPL, which promotes thromboembolism.    In paper III, a genetic locus in IRF8 was identified to be associated to coronary heart disease (CHD) in SLE. The association remained after adjustment of other CHD risk factors.  Patients with the IRF8 risk variant had increased CCA-IMT, more carotid plaques and reduced frequency of circulating B cells. Weaker binding of nuclear protein to the risk allele was demonstrated, suggesting a regulatory function of the IRF8 risk variant.    In paper IV, activated T cells were found to strongly enhance the IFNα production by pDC stimulated with RNA-containing immune complexes via GM-CSF and IL-3. Activated SLE T cells enhanced the IFNα production to the same extent as T cells from healthy controls. This finding together with previous observations in SLE of increased levels of GM-CSF and IL-3 suggests that T cells contribute to the activated type I interferon system in SLE.    In conclusion, this thesis demonstrates that genetic predisposition is important for CVD in SLE and describes a new role for T cells in the pathogenesis of SLE.

Dendritic cells (DC) are antigen-presenting cells that are critical for the initiation and regulation of the immune response. Several DC subtypes within mouse spleen have previously been characterised and these include the plasmacytoid (pDC), and conventional DC (cDC) of the CD8+ and CD8- subtypes. Each subtype appears to have a specialised role in the various arms of immunity and tolerance. Less clear is the process by which these DC develop from haematopoietic precursors, of the precursor stages and branch points from bone marrow (BM) stem cells to each of the peripheral DC subtypes. The research described herein had the aim of identifying and isolating some of the intermediate precursors of DC, downstream of stem cells, and determining whether these differed in the steady-state versus inflammation. Particular was given to DC of the spleen. Experiments that sought the identity of such precursors involved both i) transfer of cell fractions that contained DC precursors into steady-state or inflamed recipient mice to assess their in vivo development at later times, and ii) analysis of an in vitro culture system to question whether it reflected development of the steady-state DC subtypes.

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

The cytokines GM-CSF, IL-3 and IL-5 are central regulators of haemopoietic cell functions and are pivotal in the regulation of haemopoiesis and inflammatory responses of myeloid cells. In particular, these cytokines have been shown to perform essential functions in host defence against foreign pathogens through their ability to regulate innate immune responses in myeloid cells. As key regulators of such important processes, these cytokines play an important role in human inflammatory pathologies such as rheumatoid arthritis, asthma, multiple sclerosis and psoriasis as well as a number of leukemias such as JML and CMML. GM-CSF, IL-3 and IL-5 signal through receptors containing α subunits specific to each cytokine and a common β subunit (βc). Cytokine stimulation leads to tyrosine phosphorylation of the βc and promotes specific responses such as proliferation, survival and activation of haemopoietic cells. Mouse knockout studies identified a key function of these cytokines in the activation of effector functions of myeloid cells, including production of reactive oxygen species (ROS) and phagocytosis. These earlier studies provide a link between cytokine signalling and inflammation, but the molecular mechanisms by which βc activation regulates effector cell functions, and the receptor motifs involved, are unknown. The aim of this thesis was to address two broad questions with regard to βc signalling: (1) Does βc regulate specific cellular responses by phosphotyrosine-independent mechanisms? (2) What are the molecular mechanisms by which βc initiates signalling to promote specific biological responses such as activation of effector cell functions? To address the first question, we have focussed on Serine 585, a potential 14-3-3 binding site which lies in the cytoplasmic potion of huβc. Out results show that the mutation huβc S585G disrupted the interaction of 14-3-3ζ with βc, whilst not affecting receptor tyrosine phosphorylation. Both mouse and human βc were shown to interact with 14-3-3 proteins, indicating that this interaction is conserved between these species. Significantly, a huβc S585G mutant was unable to promote haemopoietic cell survival in response to IL-3. These results identify a new mechanism by which cytokine receptors are able to couple to downstream signalling pathways that regulate cell survival. An approach was developed and optimised to analyse specific GM-CSF-mediated responses in monocytes/macrophages expressing wildtype or mutant huβc, (including huβc S585G that was defective in regulating survival). Bone marrow-derived muβc -/-;muβIL-3 -/- monocytes/macrophages were retrovirally transduced with constructs expressing wildtype or mutant huβc, along with huGMRα, then purified by FACS. Two assays were established to measure effector functions in the transduced monocyte/macrophages; (1) a flow cytometry assay for ROS production, and (2) an assay for phagocytosis. The capacity for GM-CSF to prime (i.e. enhance effector functions) ROS production and phagocytosis was investigated in huGMRα-transduced monocytes/macrophages. Our results have identified two key residues in the cytoplasmic domain of βc subunit: Tyrosine 577 (required for huβc interaction with the adaptor protein Shc) and serine 585 (required for 14-3-3 association), that are essential for the ability of GM-CSF to regulate key effector functions in monocytes/macrophages. These novel findings are significant in that they establish a molecular link between the GM-CSF/IL-3/IL-5 receptor and the regulation of both haemopoietic cell survival and inflammatory responses, and therefore have important implications in our understanding of inflammatory diseases such as rheumatoid arthritis and asthma. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1317007 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

The fetus and its placenta, collectively called the conceptus, are semi-allogeneic to the mother, as they express transplantation antigens of paternal origin. Foreign tissues generally experience immunological rejection by the host immune system; however in a normal healthy pregnancy the conceptus does not undergo immune attack. Emerging evidence indicates the conceptus avoids rejection through a number of mechanisms including the induction of active maternal immune tolerance specific for paternal antigens. However, the mechanisms responsible for establishing this tolerance remain undefined, including the timing of the first encounter with paternal antigen and the cellular processes by which paternal antigen is presented to the maternal immune system. Exposure to paternal transplantation antigens occurs in two waves: initially in the context of male seminal fluid at conception, and secondly after placental trophoblast invasion of maternal tissues in mid-gestation pregnancy. Therefore the aim of this research was to evaluate the female immune response to paternal antigens in seminal fluid and those associated with the conceptus. The mechanisms of antigen presentation, the impact of the cytokine environment and the consequences of T cell activation on pregnancy were also investigated. A transgenic system using ovalbumin (OVA) as the model paternal antigen was established. The transgenic Act-mOVA mouse expresses OVA constitutively and ubiquitously under a B-actin promoter and OVA was shown to be present in seminal fluid and in the fetal and placental tissue of sired progeny. The OVA-reactive CD8+ OT-I and CD4+ OT-II T cells were employed to gauge the relative amount of OVA antigen presented, with the strength of the maternal immune response quantified based upon the extent of T cell proliferation, as assessed by CFSE dye-dilution. Utilising bone marrow chimeric mice, it was demonstrated that upon insemination by an Act-mOVA male, seminal fluid-derived OVA was processed and indirectly presented by maternal bone marrow-derived antigen presenting cells to induce activation and proliferation of the CD8+ OT-I T cells within the uterinedraining para-aortic lymph nodes of the female. Likewise, OT-II T cells were responsive to MHC class II-restricted presentation of seminal fluid OVA. Post-implantation conceptus-derived OVA was detected within peripheral lymph nodes and the spleen where it was presented via the MHC class I and class II-restricted pathways to induce systemic proliferation of both OT-I and OT-II T cells. Furthermore, as gestation advanced the extent of OVA presentation and hence T cell proliferation intensified. Conceptus-derived OVA was still presented systemically until 20 days pp. The impact of the uterine cytokine environment was assessed to determine its influence on seminal OVA antigen processing and presentation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a key factor in regulating the leukocyte population of the female reproductive tract. GM-CSF-deficient female mice were unable to process and present seminal fluid OVA as effectively or efficiently as their wildtype counterparts, as assessed by their reduced capacity to drive OT-I and OT-II T cell proliferation following insemination by an Act-mOVA male. Finally, with highly-reactive OVA-specific T cells activated in response to seminal and conceptus OVA antigen, it was of interest to determine the effect of OT-I T cell activation on fetal survival and pregnancy success. It was found that OT-I T cells activated in vivo to paternal OVA antigen in the context of seminal fluid and pregnancy were not deleterious to pregnancy outcomes. However the transfer of cytotoxic OT-I T cells generated in vitro in the presence of an IL-2 into female mice carrying OVA-expressing conceptuses was detrimental to fetal survival. Collectively these experiments demonstrated that the initial exposure to paternal antigen, and hence the first opportunity to develop paternal antigen-specific tolerance, occurs at insemination. Paternal antigen is presented to the maternal T cell repertoire throughout gestation and may play a role in maintaining immune tolerance during pregnancy. The processing and presentation of paternal-derived antigen is chiefly performed by female bone marrow-derived antigen presenting cells. The cytokine environment of the mated female reproductive tract is critical in allowing optimal antigen processing and presentation, to generate an immune response consistent with maternal immune tolerance of the conceptus. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Allergic asthma is an immunological disease, mediated by CD4+ Th2 cells, and its prevalence has increased over recent decades. Features of allergic asthma include airway hyperresponsiveness (AHR), airway eosinophilia, excessive airway mucus production, and increased IgE and Th2 cytokine levels. Airway remodeling with pulmonary fibrosis is noted in the progress of asthma. In this thesis, a murine model of allergic asthma was used to investigate the effect of cyclophosphamide (CY) on asthma and the involvement of regulatory T cells (Treg), and the role of Granulocyte-macrophage colony stimulating-factor (GM-CSF) in allergic asthma by using GM-CSF knockout mice. CY is a cytotoxic agent, which paradoxically augments several immune responses. The first part of this thesis was aimed to study the effects of CY in a murine model of allergic airway inflammation. BALB/c mice were immunized with ovalbumin (OVA) on days 0 and 14, and challenged with aerosolized OVA from days 21 to 27. Some mice additionally received CY on days -2 and 12. In the CY-treated animals, pronounced worsening of inflammatory features was noted, including increases in eosinophil infiltration, epithelial thickness, mucus occlusion and eosinophil numbers in bronchoalveolar lavage fluid (BALF). Increased total and OVA-specific serum IgE were also noted in the CY-treated animals. In cell cultures from peritracheal lymph nodes, the Th2 cytokines IL-4 and IL-5 were elevated in animals treated with CY. It was hypothesized that the effects of CY could be caused by reduced immunosuppression mediated by Treg. mRNA expression of the immunosuppressive cytokines IL-10 and TGF-beta was reduced in the lungs of CY-treated mice. The expression of FoxP3, a marker of naturally occurring Treg, was significantly reduced in spleens, thymuses and peritracheal lymph nodes after the second injection of CY, and in the lung tissue after allergen challenge in CY-treated mice. Furthermore, lung IL-10-producing CD4+ T cells and CTLA-4+-bearing CD4+ T cells were reduced after allergen aerosol challenge in CY-treated mice. Thus CY worsened the features of allergic pulmonary inflammation in this model, in association with increased production of IgE and Th2 cytokines. The reduction in expression of FoxP3 and immunosuppressive cytokines by CY suggests that toxicity to Treg may contribute to the increased inflammation. GM-CSF plays a role in the growth, development, and maturation of bone marrow hemopoietic cells into mature blood cells, and has been proposed to be involved in potentiating the function of inflammatory cells in allergic inflammation. In the second part of this thesis, GM-CSF knockout (KO) mice were used to investigate the role of GM-CSF. In allergic KO mice, airway eosinophils were only shown in the perivascular, but not peribronchial areas in the lung, compared to the allergic wild-type (WT) mice in which eosinophil infiltration appeared in both areas. Eosinophil numbers were drastically reduced in the bronchoalveolar lavage fluid (BALF) of KO mice. IL-5 production in the lung tissue and BALF in allergic KO mice was reduced; similar results were also found in peritracheal draining lymph nodes after in vitro stimulation assays. However, IL-4 and IL-13 production, airway hyperresponsiveness (AHR), and serum IgE production were not affected in allergic KO mice. Surprisingly, lung IFN-gamma mRNA and BALF levels were increased in allergic KO mice. Lung mRNA levels of CCR3, a key chemokine receptor on eosinophils, were significantly reduced in allergic KO mice, whereas expression of the chemokines eotaxin and RANTES were at similar levels in allergic KO and WT mice. Lung mRNA levels of the IFN-gamma-inducible chemokines Mig (CXCL9) and IP-10 (CXCL10), which are antagonists of CCR3, and their receptor CXCR3 were increased in allergic KO mice, compared with allergic WT mice. Data obtained from flow cytometry showed more eosinophils survived in the lung of WT mice than KO mice. Another allergy model, a peritoneal allergy model was performed to investigate inflammation in a different model. Leukocyte subpopulations such as neutrophils, eosinophils, macrophages, and lymphocytes were reduced in the peritoneal lavage fluid of allergic KO mice. The findings revealed that GM-CSF is essential for IL-5 production, pulmonary airway eosinophilia and eosinophil survival. In the absence of GM-CSF, over-production of IFN-???? may induce chemokines, including Mig and IP-10, which are antagonists for CCR3 and may reduce airway eosinophil infiltration. In this thesis, a murine model of allergic asthma has been used to obtain novel findings on the regulation of allergic inflammation. The results with CY are relevant to the treatment of asthma patients with CY and other cytotoxic agents. The findings in the GM-CSF KO mice suggest that GM-CSF is a potential therapeutic target in asthma, and that in assessment of new therapeutic agents for asthma, effects on GM-CSF should be considered.

Mémoire numérisé par la Direction des bibliothèques de l’Université de Montréal / Notre système hématopoïétique est un système de développement dynamique dont le rôle est de maintenir un niveau suffisant de cellules sanguines fonctionnelles afin d'assurer la survie de notre organisme. C'est ainsi que le système hématopoïétique donne lieu aux huit différentes lignées cellulaires connues. Les cellules sanguines différenciées incluent les erythrocytes ou globules rouges, les mégakaryocytes qui donneront lieu aux plaquettes, les granulocytes et macrophages, les basophiles et eosinophiles, ainsi que les cellules lymphoïdes B et T. Tous trouvent leur origine à partir d'une population de cellules souches hématopoïétiques. La différenciation de cellules hématopoïétiques primitives en granulocytes et monocytes matures constitue un processus de développement complex appelé la myélopoïèse. Le développement de ces cellules primitives en monocytes et granulocytes fonctionnels est en partie contrôlé par une myriade de facteurs de transcription dont le rôle est d'activer l'expression de gènes essentiels à une ou plusieurs lignées. Ces facteurs de transcription peuvent agir de façon positive ou négative à la régulation de gènes codant entre autres pour des facteurs de croissance, leurs récepteurs, pour des molécules d'adhésion, pour des enzymes ou bien pour d'autres facteurs de transcription. 0 Le développement de cellules myéloïdes primitives et de leur différenciation subséquente en cellules myéloïdes matures est également contrôlé par un vaste réseau de facteurs de croissance hématopoïétiques. Tout comme les facteurs de transcription, ces facteurs de croissance, ou cytokines, peuvent être spécifiques à une lignée particulière ou bien peuvent avoir une x n contribution plus générale. Par exemple, le G-CSF (granulocyte-colonystimulating factor) est un facteur de croissance essentiel au développement granulocytaire alors que l'IL-3 (interleukin-3) soutient la prolifération et la survie de plusieurs lignées. Quelle est l'importance d'étudier un processus tel que la myélopoïèse? Environ 120 millions de nouveaux neutrophiles sont générés chaque jour chez l'adulte normal. Ces neutrophiles jouent un rôle fondamental dans notre système de défense. En effet, les neutrophiles agissent comme première ligne de défense face à l'invasion de particules étrangères. Ils participent à notre système immunitaire inné principalement par leur action phagocytaire. Les macrophages quant à eux, jouent un rôle similaire c'est-à-dire qu'ils participent également à notre système immunitaire inné en éliminant toutes particules étrangères par phagocytose. Cependant, ils possèdent une caractéristique additionnelle. Ils jouent également un rôle dans notre système immunitaire adaptif en agissant comme cellules présentatrices d'antigènes aux différentes cellules lymphoïdes T. Chaque cellule est exposée à une multitude de signaux présents dans son environnement. La cellule se doit d'être en mesure de reconnaître et de répondre de façon spécifique à cette panoplie de signaux externes. Elle accompli ceci de plusieurs façons. Premièrement, afin de répondre à un signal particulier, la cellule se doit d'exprimer le récepteur spécifique au signal reçu. Ensuite, elle doit être en mesure d'intégrer et d'interpréter ce signal afin de le transformer en une réponse biologique concrète. Pour cela, la cellule doit posséder la machinerie interne nécessaire à la traduction et la transformation du signal. Les facteurs de croissance sont un exemple de signal externe présent dans l'environnement d'une cellule hématopoïétique. Ces facteurs de croissance sont connus pour être impliqués dans la régulation de la prolifération et de la survie cellulaire. Ces facteurs se lient à leurs récepteurs appropriés exprimés à la surface de certaines cellules. Généralement, cette liaison induit l'activité catalytique du récepteur qui donne suite à l'activation de plusieurs voies de signalisation. Ces différentes voies de signalisation ciblent différents facteurs de transcription qui sont associés à différentes réponses biologiques correspondantes. Ces facteurs de transcription, dont l'activité est induite par l'induction de voies de signalisation spécifique, vont être responsables du contrôle de 1'expression de gènes essentiels à la détermination du destin cellulaire. Vu l'importance de réguler de façon étroite l'expression de tels gènes, l'activité des facteurs de transcription se doit d'être sous haute surveillance. Le temps d'expression ainsi que l'endroit d'activation des facteurs de transcription deviennent donc des variables importantes. Un facteur de transcription responsable de la destinée d'une cellule se doit d'être exprimé dans la bonne cellule et d'être actif au bon moment dans le processus de développement de cette cellule. Souvent, l'activité et l'expression de ces facteurs de transcription sont contrôlés par des facteurs externes tels que les facteurs de croissance. Un modèle qui se veut de plus en plus populaire, malgré qu'aucun exemple définitif n'ait été encore démontré, suggérerait qu'un signal externe serait en mesure d'affecter directement l'activité d'un facteur de transcription qui lui se voudrait essentiel à la détermination de la destinée cellulaire. Le facteur de transcription myélo-spécifique PU.1 est un membre de la famille Ets. Il fût découvert simultanément de deux façons différentes. D'un xii n côté, il fût identifié à partir d'une librairie de cDNA de macrophages comme étant un facteur pouvant lier avec haute affinité, le promoteur du gène MHC classe II 1-Ab. D'autre part, il fût identifié comme site d'intégration préféré du provirus SFFV (Spleen Focus Forming Vims), un provirus associé à une érythroleucémie induite par le virus Friend. Cependant, il est important de mentionner que PU. l n'a pas été impliqué dans l'induction de leucémies humaines en partie dû au fait que son locus ne correspond pas à un site fréquent de translocation chromosomique. La famille de facteurs de transcription Ets est caractérisée par son domaine de liaison à l'ADN (domaine Ets). Ce domaine basique situé en C-terminal lie des séquences riches en purines GGAA/T dénommées boîtes PU. Ets-1, Ets-2 et Spi-B sont d'autres exemples de facteurs appartenant à cette grande famille. PU.1 est un facteur de transcription exclusif au système hématopoïétique. Son rôle dans le développement hématopoïétique semble assez bien décrit. La suppression complète du gène est associée à une absence complète de macrophages et de cellules B matures. Par contre, des monocytes et des précurseurs de cellules B sont présents mais en nombre réduit. Il y a également présence de granulocytes mais au développement et au fonctionnement anormal. Les cellules érythrocytaires ne sont, quant à elles, pas affectées. Ces résultats démontrent bien le rôle primordial de PU.1 dans le développement des cellules B ainsi que dans le développement myéloïde. Cela en fait un excellent candidat comme déterminant génétique dans rengagement du destin cellulaire. Le GM-CSF (granulocyte-macrophage colony-stimulating factor) est un facteur de croissance hématopoïétique impliqué dans la prolifération, la survie et le développement de cellules primitives hématopoïétiques avec des effets additionnels importants sur le développement myéloïde. C'est justement par son implication déterminante dans le développement des cellules granulocytaires et monocytaires que le GM-CSF est de plus en plus utilisé à des fins cliniques. Le GM-CSF fait partie de la grande famille des cytokines qui comprend entre autres l'Epo, Steel Factor, l'IL-3 et le G-CFS. Le GMCSF lie son récepteur qui est exprimé spécifiquement à la surface de certaines cellules et cette liaison active différentes voies de signalisation qui se traduisent par différentes réponses biologiques. Le récepteur du GM-CSF appartient à la super famille des récepteurs de cytokines. Il est composé d'une chaîne a et d'une chaîne p. La chaîne a est spécifique au récepteur et lui confère une basse affinité de liaison à son ligand. La chaîne P est commune à d'autres récepteurs tels les récepteurs de l'IL-3 et de l'IL-5. Elle ne possède aucune capacité de liaison. Par contre, en s'associant à la chaîne a, elle confère à celle-ci, une haute affinité de liaison au GM-CSF. De plus, c'est la chaîne R qui est principalement responsable d'activer les différentes voies de signalisation. En effet, son domaine cytoplasmique comporte de nombreux résidus de tyrosines responsables du recrutement de différents facteurs, ainsi que de deux boîtes situées à proximité du domaine membranaire qui sont responsables du recrutement de Jak2, le facteur actif dans le complexe. Les voies de signalisation qui découlent de l'activation du récepteur par son ligand sont bien décrites. Il est connu que certaines voies conservées, telles les voies MAPK, Jak-STAT et PI3K, sont activées suite à l'activation du récepteur et que ces voies sont impliquées dans des réponses de survie et de prolifération myéloïde. Par centre, rien n'est bien établi en ce qui concerne la différenciation myéloïde. Aucun facteur de transcription impliqué directement dans la détermination du destin cellulaire n'a été identifié clairement. Bien que l'on connaisse des exemples de facteurs responsables d'activer des gènes qui eux, sont spécifiques à une lignée, très peu d'informations concernant les événements transcriptionnels conduisant à la détermination du destin cellulaire sont connues. Le rôle de PU.1 dans le développement myéloïde est bien établi. Cependant, jusqu'à quel point PU.1 est-il déterminant pour qu'une cellule primitive prenne la décision de s'engager de façon irréversible vers une destinée myéloïde? L'importance de PU.1 et du GM-CSF dans le développement myéloïde a été clairement, mais indépendamment démontrée. Cependant, il n'existe aucune documentation démontrant le lien possible entre ces deux facteurs dans le développement myéloïde. Dans cette étude, nous démontrons clairement que le facteur de transcription myélo-spécifique PU. l agit comme effecteur du signal par le GM-CSF dans le développement myéloïde. En d'autres mots, l'activation de PU. l et son effet déterminant dans le développement myéloïde sont directement liés à l'activation du récepteur du GM-CSF par son ligand.

Dans les conditions physiologiques des robustes mécanismes immunologiques empêchent le développement des réponses exagérées aux antigènes alimentaires. En revanche, dans le cas de maladie céliaque, qui affecte environ 1% de la population occidentale, l’exposition au gluten alimentaire d’individus génétiquement prédisposés HLA-DQ2.5/DQ8 provoque l’entéropathie chronique de l’intestin grêle. Les études précédentes chez l’homme ont établi le rôle crucial de la réponse cellulaire T CD4+ restreinte par HLA-DQ2.5/DQ8 et spécifique du gluten. La réponse T CD4+ est nécessaire mais cependant insuffisante pour induire des lésions tissulaires. D’autres études ont suggéré le rôle de l’interleukine 15 (IL-15). Ainsi, l’IL-15 surexprimée dans la muqueuse des patients céliaques peut interférer avec les mécanismes d’immunorégulation et stimuler l’activation des lymphocytes intraépithéliaux T CD8+ cytotoxiques probablement induisant des lésions épithéliales. Comment les cellules T CD4+ spécifiques du gluten et l’IL-15 interagissent pour activer les lymphocytes intraépithéliaux T CD8+ et induisent des lésions n’a pas été toutefois établi. Pour répondre à cette question, nous avons créé un modèle murin basé en croisant des souris OTII possédant des cellules T CD4+ spécifiques de l’antigène modèle, ovalbumine, avec les souris transgéniques hétérozygotes surexprimant une forme secrétée de l’IL-15 humaine dans l’épithélium intestinale (souris hIL-15Tge). Les souris obtenues OTII+/- B6 and OTII+/- hIL-15Tge+/- ont été mises au régime riche en ovalbumine depuis la période prénatale jusqu’à l’âge de 3 mois. Les souris OTII+/- hIL-15Tge+/-, contrairement aux souris OTII+/- B6, exposées de façon chronique à l’ovalbumine ont développé un retard de croissance et une atrophie villositaire associée à l’expansion des cellules intestinales T CD8+ cytotoxiques, comme dans la maladie céliaque. En outre, nous avons démontré que l’IL-15 altérait l’immunorégulation par les cellules T FoxpP3+ et coopérait avec l’IL-2, produite par les cellules T CD4+ activées par l’OVA, pour l’expansion des cellules T CD8+ non-spécifiques de l’OVA. Nous suggérons que le scénario similaire pourrait opérer dans la maladie céliaque. Au cours de cette étude, j’ai observé que la surexpression chronique de l’IL-15 était associée avec l’expansion de cellules dendritiques CD103+CD11c+CD11b-. Dans la partie de résultats supplémentaires, j’ai démontré que cet effet dépend de la production de la cytokine GM-CSF secrétée par les cellules Natural Killer (NK) activées par l’IL-15 et que ces cellules dendritiques étaient enrichies en cellules CD103+ ayant une capacité accrue de cross-présentation in vitro. Ces derniers résultats illustrent comment l’IL-15 peut moduler les réponses immunes adaptatives en orchestrant la coopération entre les cellules NK et les phagocytes mononucléaires. / In physiological conditions, robust immunological mechanisms avoid adverse responses to food antigens. In contrast, in celiac disease that affects about 1% of Western populations, exposure to dietary gluten of genetically predisposed HLA-DQ2.5/ DQ8 individuals triggers a chronic small intestinal enteropathy. Previous studies in humans have established the crucial role of HLA-DQ2/DQ8 restricted gluten-specific intestinal CD4 T cell response. This CD4 T cell response is necessary but is however not sufficient to induce tissue damage. Other studies have pointed to the role of interleukin 15 (IL-15). Thus, IL-15 over-expressed in the mucosa of celiac patients can interfere with immunoregulatory mechanisms and stimulate the activation of cytotoxic CD8 T intraepithelial lymphocytes, thought to induce epithelial lesions. Whether and how gluten-specific CD4 T cells and IL-15 interact to activate CD8 T intraepithelial lymphocytes and to drive intestinal tissue damage has not been however established. To address this question, we have set up a mouse model based on the breeding of OTII mice possessing CD4 T cells specific of a model antigen, ovalbumin, with heterozygous transgenic mice overexpressing a secreted form of human IL-15 in intestinal epithelium (hIL-15Tge mice). Resulting OTII+/- B6 and OTII+/- hIL-15Tge+/- mice were exposed to dietary ovalbumin from the prenatal period until 3 months of age. Upon chronic exposure to ovalbumin, OTII+/- hIL-15Tge+ mice, contrary to their OTII+/- B6 littermates, developed growth retardation, and villous atrophy associated with expansion of intestinal cytotoxic CD8 T cells, as in celiac disease. Moreover, we showed that IL-15 impaired immunoregulation by FoxP3+ T cells and cooperated with IL-2 produced by OVA-activated CD4 T cells to stimulate the expansion of non-cognate cytotoxic CD8 T cells. We suggest that a comparable scenario can operate in celiac disease. During this study, I observed that chronic overexpression of IL-15 was associated with an expansion of CD103+CD11c+CD11b- mononuclear cells. In the Supplementary results, I have shown that this effect depends on the production of GM-CSF secreted by IL-15-activated NK cells and that CD11c+ DCs differentiated in mice overexpressing IL-15 were enriched in CD103+ cells and displayed enhanced cross-presentation abilities in vitro. The latter results illustrate how IL-15, by orchestrating a crosstalk between NK cells and mononuclear phagocytes, can modulate adaptive immune responses.

Doctor of Philosophy (PhD) / Asthma is a chronic inflammatory disease of the airways. Corticosteroid medication is the most effective currently available treatment. Complications of corticosteroid therapy are dose-dependent, however, the clinical efficacy of varying doses of inhaled corticosteroids has been studied with mixed results. A randomized, double-blind, parallel group study was used to evaluate the inhaled corticosteroid dose-response relationship for clinical endpoints and in vitro parameters of underlying airway inflammation and remodelling. The mannitol provocation test with Forced Oscillation Technique (FOT) was used to derive potential dose-differentiating endpoints. In vitro inflammatory markers were measured in alveolar macrophages from bronchoalveolar lavage. Basement membrane thickness was measured from bronchial biopsies. Eleven nonasthmatic subjects were enrolled for comparison. This thesis addresses the null hypothesis that there is no significant difference in clinical and biological effects between low dose (200mcg/day, n=11) and high dose (1000mcg/day, n=11) treatment (for 6-7 weeks) with inhaled fluticasone propionate (FP) for a range of clinical outcomes and in vitro markers of airway inflammation and remodelling. Significant changes after FP included increased FEV1, reduced airway hyperresponsiveness (AHR) (by FOT and FEV1), exhaled nitric oxide and Juniper symptom score. In addition, significant reductions occurred in expression of GM-CSF, TNF-alpha and IL-1ra in macrophages. A lower baseline FOT-derived respiratory system conductance was predictive of a greater degree of improvement in symptoms. No statistically significant differences in the changes after treatment between low and high dose FP were found in spirometry, exhaled nitric oxide, symptom scores, AHR, alveolar macrophage cytokine levels (GM-CSF, TNF-alpha, IL-1ra, IL-10) and basement membrane thickness, although there were trends towards greater improvements in many of the parameters after high dose FP. Basement membrane thickness appeared to be reduced by high dose FP, although this reduction was not statistically significant. There was a weak, but statistically significant, negative correlation between basement membrane thickness and FOT-derived conductance (r2=0.135, p=0.042). With the recognition of the limitations in the interpretation of these data, the results suggest that, in previously steroid naïve mild to moderate asthmatics, there may be only minimal benefit derived from an additional 800µg/day of inhaled fluticasone above the low dose of 200µg/day.